T lymphocytes (T cells) contact with antigen-presenting B cells initiates an activation cascade which includes an increase in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. Although T-cell/B-cell physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. Novel optical techniques at the LAMMP facility provide insights into these puzzles at the single-cell level. In this project, we study biophysical requirements for T-cell activation using an optical trap to control the orientation of T-cell/B-cell pairs and fluorescent microscopy to measure subsequent T-cell intracellular calcium level ([Ca2+]i) response. Our specific aim is to develop an optical trapping-based high resolution fluorescence imaging system (CATS) to study1) Spatial requirements for T-cell activation and relationships between stimulus intensity and response pattern (response percentage, response latency, calcium signal pattern, shape and motility changes), 2) The role of adhesion/co-stimulatory molecules, such as CD45 molecule, in T cell activation, 3) The role of actin cytoskeleton in T cell activation.